Heterogeneous assays, in general, involve determination of analytes in samples via a phase change which occurs at some time during the assay run. In a preferred form of assay, a test apparatus, such as a bibulous paper or film based strip, is contacted by the sample to be assayed. The test strip contains a component which interacts in some way with the analyte in the sample, if the analyte is present. As the sample is fluid, it diffuses or passes through the test apparatus, carrying with it any of the analyte, component, and any product resulting from interaction between analyte and component.
Usually, the interaction of analyte and component leads to formation of a reaction product, frequently a complex of analyte and component. The component carries some signal generating means, or label, which allows one to identify and/or to quantify it. The task of the investigator is to determine if the component has complexed to analyte, and/or how much has complexed. This is accomplished by measuring the label.
One fact of assays involving labeled components is that the label is carried by the labeled component regardless of whether it is bound or complexed to analyte. It therefore becomes imperative to have some way of discriminating complexed label from uncomplexed material.
This is achieved in heterogeneous assays by providing a solid phase bound moiety which discriminates between complexed and uncomplexed labeled components. The solid phase bound moiety permits separation of phase (hence the term "heterogeneous"), and identification and quantification of complexed and uncomplexed labeled component.
Various forms of heterogeneous assays will be familiar to the skilled artisan, such as immunoenzymometric assays, which involve formation of complexes between analyte and labeled component, where the complex remains in solution and the uncomplexed label is removed from solution via a solid phase bound reaction partner; so-called "sandwich assays", where a binding partner which is labeled, complexes with the analyte followed by contact with an immobilized moiety which binds to the analyte, forming a sandwich and leaving uncomplexed label in the liquid phase. These two general examples are certainly not exhaustive of the various forms of assays which can be performed.
Many materials are available as the choice of label. One particularly preferred class of labels is enzymes. When an enzyme is used as the label, the identification of complex involves contacting the label, once complexes have formed, with a substrate which reacts with the enzyme to give a detectable signal. Frequently, although not exclusively, the signal is visually determinable, such as the formation of a color or a color change.
While the enzyme label type of assay is quite useful, there are problems with it. One serious problem, which is now discussed, is the problem addressed by this invention.
Generally, the substrate with which the enzyme label reacts is incorporated into the test apparatus together with the label. While these are usually incorporated in the device at different points, a test device is usually absorbent over its entire reactive surface, as it must be for the various components of the test system to be brought together in functional relationships. If the substrate is brought into contact with the labeled component before the desired reaction takes place, the detectable signal is generated at an inappropriate time. When this happens, the test results' accuracy cannot be guaranteed, and, at times, the results cannot even be determined at all. While controls can be run to give baseline or zero values, it must be understood that in practice, the number of heterogeneous assays which are run over any given period is staggering. Performing a control run for each of these thus becomes a theoretical possibility, but a practical impossibility. Surprisingly, the art shows that little attention has been paid to this particular problem. Calenoff, et al., U.S. Pat. No. 4,528,267, teach inhibition of reactions between IgE and allergens, and Monji, et al., U.S. Pat. No. 4,323,647 teach the general principle that there are competitive and non-competitive inhibitors for enzymes, as well as an assay which involves the use of an antibody as an inhibitor of a complex of antigen and enzyme. In this system, one is determining an antigen in a sample. The labeled antigen is identical to that being determined, and the antibody binds to both labeled antigen and sample antigen. Labeled and unlabeled antigen compete for antibody. That portion of labeled antigen which does not bind to antibody is free to bind to solid phase bound substrate, while antibody bound antigen-enzyme complex is sterically hindered from doing the same. By this rather involved process, one determines the amount of antigen present in the sample. It will be understood, that this system could not be used to address the problem referred to supra, because the enzyme label is still free to react with substrate, should premature contact take place.
European Patent Application 202 081 teaches use of an alternate substrate to delay reaction of the enzyme in the test device with the substrate which will be used to form the detectable moiety. The alternate substrate, however, is chosen so that it is acted upon by the enzyme: expressed another way, the inhibition of the enzyme lasts only as long as the substrate itself remains undigested. This differs from a true inhibitor, which will impede the enzyme until actually removed.
Hence it is an object of the invention to provide a method for inhibiting formation of a detectable signal produced by interaction of enzyme label and substrate until the desired reaction between analyte and receptor occurs, wherein the removal of the inhibitor lies in the control of the investigator.
It is further object of the invention to provide a test apparatus which can be used in connection with the method outlined above.
It is still another object of the invention to provide a reagent or kit which can be used to prepare a device for determination of an analyte, utilizing the method described above.
How these and other objects of the invention are accomplished will be seen from the disclosure which follows.